1. Field of the Invention
The field of this invention is knock-in cell-based drug screens for regulators of targeted gene expression.
2. Background
Transcriptional regulation provides an ideal target for therapeutic intervention. A number of techniques are available for screening for drugs active at the level of gene transcription. Including in vitro assays such as binding assays (e.g. U.S. Pat. No. 5,563,036) and RNA polymerase assays (e.g. U.S. Pat. No. 5,635,349), cell-based assays such as cotransfection assays and Northern-blot analyses.
Inherent drawbacks of these in vitro and transfection-based assays include their limited recreation or modeling of the natural transcriptional process. On the other hand, Northern-blot analyses of natural transcripts are time and resource demanding and less amendable to high-throughput drug development programs.
The invention provides methods and compositions for screening for agents which regulate the level of targeted gene expression in a natural context. Such agents find use in modulating a wide variety of physiological manifestations of gene expression.
The subject assays are cell-based and generally involve contacting a mammalian cell comprising a mutant of a native allele encoding a reporter of the targeted gene expression, wherein the expression of the reporter is under the control of the native gene expression regulatory sequences of the native targeted allele, with a candidate agent under conditions whereby but for the presence of the agent, the reporter is expressed at a first expression level; and, measuring the expression of the reporter to obtain a second expression level, wherein a difference between the first and second expression levels indicates that the candidate agent modulates the expression of the targeted gene.
The mutant generally results from replacement of a portion of the native allele with a sequence encoding the reporter. For example, the cell may be a progeny of, a clone or, or genetically identical to a genetic knock-in cell made by homologous recombination of the native allele with a transgene comprising a sequence encoding the reporter flanked by flanking sequences capable of effecting the homologous recombination of the transgene with the native allele, a positive selectable marker positioned inside the flanking sequences and optionally, a negative selectable marker positioned outside the flanking sequences. The cell may be a primary cell residing in or isolated from an animal transgenic in the mutant or derive from a cultured cell line transgenic in the mutant.
The invention also encompasses mammalian cells and mammals transgenic in a mutant of a native allele encoding a reporter of gene expression, wherein the expression of the reporter is under the control of the gene expression regulatory sequences of the native allele, genetic knock in vectors for making such animals and cells and methods of making and using such vectors, cells and animals.